The establishment of human pluripotential stem cell lines has led to a resurgence of interest in stem cell biology. This interest is largely due to the therapeutic potential of stem cells for repairing degenerative diseases and injuries. Before recent breakthroughs in studies of human stem cells can be effectively and safely applied in the clinic, many basic questions about the biology of stem cells need to be addressed. These include: how is stem cell proliferation regulated in vivo to generate the appropriate number of daughter stem cells and differentiating progeny without forming tumors? How is the developmental potential of a stem cell restricted to a particular fate? How is pluripotentiality maintained and what steps lead to loss of pluripotentiality? The work proposed here will address these fundamental questions using the freshwater planarian, Schmidt Mediterranean, as a model organism for studying stem cell regulation. Planarians- classic models of regeneration experiments can regenerate entire animals from small fragments of their bodies. This remarkable plasticity is based upon a stem cell population present in the adult worm. These stem cells are used both to replace cells lost during the course of cell turnover and to regenerate missing structures when the animal is transected. Recent advances permit this classic system to be re-examined in detail at both cellular and molecular levels. Thus, environmental influences on stem cell proliferation will be studied by analyzing cell cycle kinetics in intact and regenerating planarians. These experiments will determine if wounding regulates the cell cycle of planarian stem cells. If it does, these experiments will also define the phases of the cell cycle that are regulated, an important consideration for understanding the signaling pathways that lead from wounding to proliferation. The development of automated in situ hybridization techniques and the availability of over 4000 unique planarian cDNAs provide starting material for defining stem cell-specific genes and raising monoclonal antibodies to identify and study stem cells. Finally, functional analysis using double-stranded RNA-mediated genetic interference will be used to identify genes that play critical roles in stern cell regulation.